CRISPR-mediated genome editing is accelerating basic research and its application to improve human health. In traditional genetic model organisms, like the fruit fly Drosophila melanogaster, CRISPR has accelerated and simplified genetic manipulations. In organisms where genome modifications have been more challenging, like the malaria mosquito Anopheles gambiae, CRISPR-based genome editing presents new opportunities to study their biology and to disrupt disease transmission. Here we propose to simplify and expedite CRISPR-based genome editing for fly and mosquito researchers. We propose to achieve these goals in two aims: 1) We will develop and optimize a universal CRISPR toolkit for genome editing in Drosophila. In aims 1.a and 1.b, we will optimize a set of simple-to-use targeting vectors that will speed and simplify the implementation of CRISPR-Cas9 gene editing in Drosophila. In aim 1.c., we will modify these optimized vectors to generate a set of reagents that will allow investigators to readily create fluorescently labeled targeted knockouts and reporter knock-ins (including Gal4, GFP and epitope-tags) by simply combining gene-specific gRNAs with these universal reagents. Aim #2: Develop and optimize a uHITI toolkit for genome editing in Anopheles. In aim 2.a., we will optimize our targeting approach for use in A. gambiae. In aim 2.b., we will modify these optimized A. gambiae vectors to generate a set of reagents to allow investigators to create targeted knock-outs and reporter knock-ins (including the QF transcription factor, GFP, epitope-tags and a genetically encoded calcium indicator) by combining gene-specific gRNAs with these universal reagents. These tools will simplify and expedite the generation of targeted knock-outs and knock- ins by investigators studying Drosophila and Anopheles. In Drosophila, these tools will also increase the feasibility of creating genome-wide knock-out and knock-in collections. In Anopheles, these tools can expedite the introduction of complex gene cassettes into different genomic locations in order to disrupt vector populations.